PSl/gamma-secretase is a large protease complex mainly comprised of membrane proteins responsible for intramembranous cleavage of its substrates. PS1/gamma-secretase was originally shown to cleave the amyloid precursor protein (APP) and Notch, releasing the Abeta peptide and the Notch intracellular domain, respectively. We and others have recently identified additional substrates undergoing PS1-dependent proteolytic processing. Although these substrates perform a variety of different functions, we will explore the potential relationship of the roles played by the PS1/gamma-secretase complex specifically in Abeta generation and cell-cell junctions. We have recently identified a novel substrate for PS1/gamma-secretase activity, nectin-1, an adherens junction protein involved in synapse formation, and we are currently characterizing additional substrates from cell-cell junctions. For the proposed studies, we have developed in vitro CHO cell aggregation and dissociation assays to assess the role of PS1/gamma-secretase in cell-cell junctions. We have found that overproduction of nectin-1 favors aggregation and, conversely, lowers the ability of the cells to dissociate. Inhibition of PS1/gamma-secretase activity in these cells reduces dissociation by approximately 75%, while stabilizing nectin-1 bound to the actin cytoskeleton. Similarly, PS1/gamma-secretase function is likely to regulate the turnover of its other substrates during changes in cell-cell adhesion, as already reported for E-cadherin. We hypothesize that pathological mutations in PS1 or its substrates (APP) may perturb a delicate balance in the turnover of these proteins a consequence of which is altered Abeta generation. To follow up our preliminary results and extend our studies regarding the role of PS1/gamma-secretase in cell-cell junctions and Abeta generation, we propose to address two main specific aims. First, we will assess the role of PS1/gamma-secretase activity in cell-cell junction formation and in modulating the function of cell-cell junction proteins by modulating PS1/gamma-secretase activity (e.g. by using known FAD and dominant negative mutations and gamma-secretase inhibitors) and then evaluating potential effects on the processing and subcellular localization of its substrates as well as effects on cell-cell aggregation/dissociation in vitro and synapse formation in primary neuronal cultures. Second, we will modulate cell-cell adhesion and study corresponding changes in PS1/gamma-secretase complex formation, localization and proteolysis of its substrates, paying particular attention to potential effects on APP processing and Abeta generation. The overarching goal of this application is to define the pathogenic mechanism by which FAD mutations in the PS genes lead to the onset of AD by understanding the role of PS1/gamma-secretase in the formation of cell-cell junctions and its relationship to Abeta production.